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Yamaguchi S.,Institute of Regenerative Medicine and Biofunction | Niwa R.,Bioscience Technology | Kazuki Y.,Tottori University | Ohbayashi T.,Institute of Regenerative Medicine and Biofunction
Yonago Acta Medica

Exactly controlled conditional gene expressing systems are crucial for genomic functional research, animal transgenesis and gene therapy. Bacterial artificial chromosomes (BACs) are optimal for harboring long fragments of genomic DNA or large cDNA up to 300 kb in size. Therefore, BACs are available to produce transgenic cells and animals for the functional studies of genes. However, BAC can insert DNA randomly into the host genome, possibly causing unpredicted expression. We previously developed a human artifi{ligature}cial chromosome (HAC) vector from human chromosome 21 using chromosome engineering. The HAC vector has several important characteristics desired for an ideal gene delivery vector, including stable episomal maintenance, and the ability to carry large genomic DNA containing its own regulatory element, thus allowing physiological regulation of the transgene in a manner similar to that of the native chromosome. In this study, we develop a system fusing BAC library and HAC technology together to allow tight control of gene expression This system enables BAC to be cloned into the defined locus on the HAC vector by the Cre/loxP system. In addition, the genome in the BAC is possible to be engineered freely by the BAC recombineering technology. This system is a highly efficient tool for the rapid generation of stringently controlled gene expression system on the HAC vector. Source

Otsuka S.,Tottori University | Otsuka S.,Institute of Regenerative Medicine and Biofunction | Sakamoto Y.,Tottori University | Siomi H.,Keio University | And 6 more authors.
Brain and Development

Fragile X syndrome (FXS), which is the most common form of familial mental retardation, is caused by the expansion of the CGG repeat in the FMR1 gene on the X chromosome. Previous studies have suggested that as compared to other populations, Japanese have a lower prevalence of FXS. In addition, in the normal population, there are no carriers who have the premutation allele. We analyzed a total of 946 normal Japanese (576 males and 370 females) and attempted to estimate the frequency of the FMR1 allele. Within this population, we found that 1,155 alleles were in the normal range (less than 40 CGG repeats) and had a modal number of 27 repeats (35.75%). No carriers with premutations (55-200 CGG repeats) were observed in this normal population. We also identified six intermediate-sized alleles (40-54 CGG repeats), with a reported incidence of 1 in 103 males and 1 in 324 females. However, this allele frequency was different from that previously reported for the Japanese population. Since data from previous studies has suggested that FXS might possibly be associated with the genetic mechanism of autism, we also analyzed the length of the CGG repeats in 109 autistic patients. In all cases the CGG repeat numbers were within the normal range (16-36 repeats) and no individuals presented with expanded premutation or intermediate alleles. This finding indicates that the length of the CGG repeat within the FMR1 is unlikely to be responsible for autism in Japanese. © 2008 Elsevier B.V. All rights reserved. Source

Miyazaki S.,Nojima Hospital | Hamada T.,Tottori University | Hirata S.,Hirata Clinic of Internal Medicine | Ohtahara A.,Sanin Rosai Hospital | And 13 more authors.
Clinical and Experimental Hypertension

Purpose: To examine effects of a long-acting calcium channel blocker (CCB) azelnidipine on uric acid metabolism in hypertensive patients. Methods: Azelnidipine was administered to 72 patients at a daily dose of 8omg or 16omg. In 22 cases out of the 72 patients, a different CCB was switched to azelnidipine. Blood pressure was measured and biochemical parameters of blood and urine were evaluated before and 2-3 months after the administration. Results: Azelnidipine significantly decreased both systolic and diastolic blood pressure and the heart rate. It decreased both serum urate levels and the urinary uric acid to creatinine ratio (Uur/Ucr), but did not affect the uric acid clearance to creatinine clearance ratio (Cur/Ccr). Azelnidipine decreased both Uur/Ucr and Cur/Ccr in patients with Uur/Ucr 0.5 or 0.34, although it did not change these clearance parameters in patients with Uur/Ucr <0.5 or <0.34. Azelnidipine decreased the serum urate levels and Uur/Ucr in hyperuricemic patients with uric acid levels 7.0omg/dL in males and 6.0omg/dL in females. It did not change these parameters in normouricemic patients with serum urate levels <7.0omg/dL in males and <6.0omg/dL in females. Azelnidipine decreased Uur/Ucr and Cur/Ccr in hyperuricemic patients with normal or overexcretion of uric acid, although it did not change these clearance parameters in hyperuricemic patients with uric acid hypoexcretion. Conclusions: Azelnidipine decreased the serum urate acid levels and Uur/Ucr, and this response was most prominent in hyperuricemic patients or patients with normal and overexcretion of uric acid. © 2014 Informa Healthcare USA, Inc. Source

Kazuki Y.,Institute of Regenerative Medicine and Biofunction | Kazuki Y.,Tottori University | Kobayashi K.,Chiba University | Aueviriyavit S.,Chiba University | And 19 more authors.
Human Molecular Genetics

Human CYP3A is the most abundant P450 isozyme present in the human liver and small intestine, and metabolizes around 50% of medical drugs on the market. The human CYP3A subfamily comprises four members (CYP3A4, CYP3A5, CYP3A7, CYP3A43) encoded on human chromosome 7. However, transgenic mouse lines carrying the entire human CYP3A cluster have not been constructed because of limitations in conventional cloning techniques. Here, we show that the introduction of a human artificial chromosome (HAC) containing the entire genomic human CYP3A locus recapitulates tissue- and stage-specific expression of human CYP3A genes and xenobiotic metabolism in mice. About 700 kb of the entire CYP3A genomic segment was cloned into a HAC (CYP3A-HAC), and trans-chromosomic (Tc) mice carrying a single copy of germline-transmittable CYP3A-HAC were generated via a chromosome-engineering technique. The tissue- and stage-specific expression profiles of CYP3A genes were consistent with those seen in humans. We further generated mice carrying the CYP3A-HAC in the background homozygous for targeted deletion of most endogenous Cyp3a genes. In this mouse strain with 'fully humanized' CYP3A genes, the kinetics of triazolam metabolism, CYP3A-mediated mechanism-based inactivation effects and formation of fetal-specific metabolites of dehydroepiandrosterone observed in humans were well reproduced. Thus, these mice are likely to be valuable in evaluating novel drugs metabolized by CYP3A enzymes and in studying the regulation of human CYP3A gene expression. Furthermore, this system can also be used for generating Tc mice carrying other human metabolic genes. © The Author 2012. Published by Oxford University Press. All rights reserved. Source

Hashimoto M.,Chiba University | Kobayashi K.,Chiba University | Watanabe M.,Chiba University | Kazuki Y.,Institute of Regenerative Medicine and Biofunction | And 9 more authors.
Journal of Lipid Research

Here , we studied the effects of cytochrome P450 (CYP)3A deficiency on the mRNA expression of genes encoding regulators of hepatic cholesterol levels using Cyp3a - knockout ( Cyp3a-/- ) mice. The mRNA expression levels of genes encoding enzymes involved in cholesterol biosynthesis in the livers of Cyp3a-/- mice were higher than those of wild-type (WT) mice. Nuclear levels of sterol regulatory element- binding protein-2 (SREBP-2), which enhances cholesterol biosynthesis, were also higher in the livers of Cyp3a-/- mice. Binding of SREBP-2 to the Hmgcs1 gene promoter was more abundant in the livers of Cyp3a-/- mice. These results suggest that deficiency of CYP3A enzymes enhances transcription of genes encoding enzymes involved in cholesterol biosynthesis via activation of SREBP-2. On the other hand, hepatic cholesterol levels in Cyp3a-/- mice were 20% lower than those in WT mice. The mRNA expression levels of genes encoding enzymes involved in bile acid synthesis, plasma levels of 7 β -hydroxy-4-cholesten-3-one and hepatic levels of total bile acid were significantly higher in Cyp3a-/- mice than in WT mice. These findings suggest that reduction of hepatic total cholesterol in Cyp3a-/- mice would be the consequence of enhanced bile acid synthesis. Therefore, CYP3A enzymes appear to play roles in the synthesis of cholesterol and bile acid in vivo. -Hashimoto, M., K. Kobayashi, M. Watanabe, Y. Kazuki, S. Takehara, A. Inaba, S-i. Nitta, N. Senda, M. Oshimura, and K. Chiba. Knockout of mouse Cyp3a gene enhances synthesis of cholesterol and bile acid in the liver. Copyright © 2013 by the American Society for Biochemistry and Molecular Biology, Inc. Source

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